Spectrum analysis apparatus, fine particle measurement apparatus, and method and program for spectrum analysis or spectrum chart display

ABSTRACT

Provided is a spectrum analysis apparatus including a processing unit configured to generate analysis data using an analysis function in which a linear function and a logarithmic function are included as function elements and an intensity value is set as a variable from measurement data including the intensity value of light acquired by detecting the light from a measurement target object using a plurality of light-receiving elements having different detection wavelength bands.

TECHNICAL FIELD

The present technology relates to a spectrum analysis apparatus, a fineparticle measurement apparatus, and a method and program for a spectrumanalysis or a spectrum chart display. More particularly, the presenttechnology relates to a spectrum analysis apparatus and the like capableof obtaining a spectrum chart accurately reflecting opticalcharacteristics of a measurement target object.

BACKGROUND ART

A flow cytometer is an apparatus that optically measures characteristicsof fine particles by radiating light to the fine particles such ascells, beads, or the like that flow through a flow cell and detectingfluorescence, scattered light, or the like emitted from the fineparticles.

For example, when the fluorescence of cells is detected, excitationlight having an appropriate wavelength and intensity such as laser lightis radiated to a cell labeled by a fluorochrome. The fluorescenceemitted from the fluorochrome is condensed by a lens or the like, lightof an appropriate wavelength band is selected using a wavelengthselection element such as a filter or a dichroic mirror, and theselected light is detected using a light-receiving element such as aphoto multiplier tube (PMT). At this time, it is possible tosimultaneously detect and analyze fluorescence from a plurality offluorochromes labeled to cells by a plurality of combinations ofwavelength selection elements and light-receiving elements. Further, itis also possible to increase the number of analyzable fluorochromes bycombining excitation light of a plurality of wavelengths.

In the related art, analysis data of the flow cytometer is displayed bya histogram or a two-dimensional (2D) plot. Although a linear axis or alogarithmic axis is generally used as a coordinate axis representing anintensity value of light in the histogram and the 2D plot, technologyusing a biexponential axis having characteristics in which the linearaxis and the logarithmic axis are combined is also known (see NPL 1). Inthe histogram and the 2D plot using the biexponential axis as thecoordinate axis, a display of a wide dynamic range utilizingcharacteristics of the logarithmic axis is possible and simultaneously adisplay of a negative number according to characteristics of the linearaxis is also possible.

In the fluorescence detection by the flow cytometer, there is a methodof measuring an intensity of light in a continuous wavelength band as afluorescence spectrum in addition to a method of selecting a pluralityof pieces of light of a discontinuous wavelength band using a wavelengthselection element such as a filter and measuring an intensity of lightof each wavelength band. In a spectral flow cytometer in which afluorescence spectrum is measurable, the fluorescence emitted from thefine particles is spectrally separated using a spectral element such asa prism or a grating. The spectrally separated fluorescence is detectedusing a light-receiving element array in which a plurality oflight-receiving elements having different detection wavelength bands arearranged. As the light-receiving element array, a PMT array or aphotodiode array in which light-receiving elements such as PMTs orphotodiodes are arranged in one dimension or an array of a plurality ofindependent detection channels of 2D light-receiving elements such ascharge-coupled devices (CCDs) or complementarymetal-oxide-semiconductors (CMOSs) is used.

CITATION LIST Patent Literature

-   [PTL 1]-   JP 2003-83894A

Non Patent Literature

-   [NPL 1]-   A New “Logicle” Display Method Avoids Deceptive Effects of    Logarithmic Scaling for Low Signals and Compensated Data. Cytometry    Part A 69A:541-551, 2006.

SUMMARY Technical Problem

The analysis data in the spectral flow cytometer can be displayed by aspectrum chart in addition to the histogram and the 2D plot. In thespectrum chart, a channel or a detection wavelength of thelight-receiving element is represented on the horizontal axis, anintensity value of light is represented on the vertical axis, andinformation (population information) regarding the number of fineparticles (an event count or density) is represented by the gradation ofcolor, a color tone, or the like. According to the spectrum chart, it ispossible to intuitively recognize a fluorescence spectrum and populationinformation of fine particles.

In the spectrum chart, the linear axis or the logarithmic axis is usedas a coordinate axis representing an intensity value of light in therelated art. However, the chart using the logarithmic axis has alimitation in that a spectrum of fine particles having a low intensityvalue is rendered with unreasonably high dispersion, and a negativenumber is not displayed. On the other hand, there is a problem in thatit is difficult to discriminate a spectral shape of fine particleshaving a low intensity value even in the chart using the linear axis.Further, in the spectrum chart of the related art, there is no methodsuitable for displaying a spectrum by subtracting a spectrum componentderived from an intensity value (background value) detected in a controlsample such as an unlabeled cell.

It is desirable to provide technology for displaying a wide dynamicrange and a negative number and obtaining a spectrum chart appropriatelyreflecting an intensity of light generated from fine particles.

Solution to Problem

In accordance with an embodiment of the present technology, there isprovided a spectrum analysis apparatus including: a processing unitconfigured to generate analysis data using an analysis function in whicha linear function and a logarithmic function are included as functionelements and an intensity value is set as a variable from measurementdata including the intensity value of light acquired by detecting thelight from a measurement target object using a plurality oflight-receiving elements having different detection wavelength bands.

The spectrum analysis apparatus includes a display unit configured todisplay the analysis data in a spectrum chart in which one axisrepresents a value corresponding to the detection wavelength band andthe other axis represents an output value of the analysis function.

According to this spectrum chart, a wide dynamic range including anegative value can be displayed, and a spectrum that appropriatelyexpresses optical characteristics of a measurement target object can bedisplayed by suppressing dispersion.

In this spectrum analysis apparatus, the processing unit is configuredto generate the analysis data by applying a function in which the linearfunction is set as a main function element for data in which theintensity value is small among the measurement data and the logarithmicfunction is set as the main function element for data in which theintensity value is large among the measurement data as the analysisfunction.

Specifically, the processing unit can generate the analysis data byapplying a function in which the linear function is set as a mainfunction element for data in which the intensity value is less than apredetermined value among the measurement data and the logarithmicfunction is set as the main function element for data in which theintensity value is greater than the predetermined value among themeasurement data as the analysis function. In this case, the displayunit displays the spectrum chart in which the axis of the output valueis set as the logarithmic axis in a region in which the output value isgreater than the predetermined value and set as the linear axis in aregion in which the output value is less than the predetermined value.

In the spectrum analysis apparatus, it is preferable that the processingunit be configured to generate analysis data corrected using theanalysis function in which the linear function and the logarithmicfunction are included as the function elements and the intensity valueis set as the variable, after subtracting measurement data including anintensity value obtained by detecting light from a control sample usinga plurality of light-receiving elements having different detectionwavelength bands from measurement data including an intensity valueobtained by detecting light from a measurement sample using a pluralityof light-receiving elements having different detection wavelength bands,and the display unit be configured to display the corrected analysisdata in the spectrum chart.

In addition, in the spectrum analysis apparatus, the measurement datacan include the intensity value of light detected and obtained by alight-receiving element array in which a plurality of light-receivingelements having different detection wavelength bands are arranged tospectrally separate the light from a measurement target object. In thiscase, it is preferable for the processing unit to correct the intensityvalue in a detection wavelength band width of each light-receivingelement and generate the analysis data.

In the spectrum analysis apparatus in accordance with the presenttechnology, particularly, the measurement target object can be fineparticles, and the optical characteristics of the fine particles can bedisplayed in the spectrum chart. In this case, it is preferable for thedisplay unit to perform a multicolor display of the spectrum chart. Themulticolor display can be performed by displaying the spectrum chartaccording to a hue, saturation, and/or brightness reflecting frequencyinformation of the fine particles.

In addition, the spectrum analysis apparatus in accordance with thepresent technology can be configured as a spectral flow cytometer,particularly, even in the fine particle measurement apparatus.

In accordance with other embodiments of the present technology, thereare provided a spectrum analysis method including: a procedure ofgenerating analysis data using an analysis function in which a linearfunction and a logarithmic function are included as function elementsand an intensity value is set as a variable from measurement dataincluding the intensity value of light acquired by detecting the lightfrom a measurement target object using a plurality of light-receivingelements having different detection wavelength bands, and aspectrum-chart displaying method including a procedure of displaying aspectrum chart in which one axis represents a value corresponding to thedetection wavelength band and the other axis represents an output valueof the analysis function.

In accordance with still other embodiments of the present technology,there are provided a spectrum analysis program for executing: generatinganalysis data using an analysis function in which a linear function anda logarithmic function are included as function elements and anintensity value is set as a variable from measurement data including theintensity value of light acquired by detecting the light from ameasurement target object using a plurality of light-receiving elementshaving different detection wavelength bands, and a spectrum-chartdisplaying program for executing: displaying the analysis data in aspectrum chart in which one axis represents a value corresponding to thedetection wavelength band and the other axis represents an output valueof the analysis function.

In the present technology, the “fine particles” includebiologically-relevant fine particles such as cells, microorganisms, andliposomes or synthetic particles such as latex particles, gel particles,and industrial particles.

The biologically-relevant fine particles include chromosomes, liposomes,mitochondria, and organelles constituting various cells. The cellsinclude animal cells (such as blood cells) and plant cells. Themicroorganisms include bacteria such as Escherichia coli, viruses suchas tobacco mosaic viruses, and fungi such as yeast. Further, thebiologically-relevant fine particles can also includebiologically-relevant macromolecules such as nucleic acids, proteins,and complexes thereof. In addition, the industrial particles, forexample, may be organic or inorganic polymeric materials, or metals. Theorganic polymeric materials include polystyrene, styrene-divinylbenzene,polymethyl methacrylate, and the like. The inorganic polymeric materialsinclude glass, silica, magnetic materials, and the like. The metalsinclude colloidal gold, aluminum, and the like. In general, shapes ofthese fine particles are commonly spherical, but may be non-spherical.In addition, a size, mass, and the like are not particularly limited.

Advantageous Effects of Invention

In accordance with the present technology, technology for displaying awide dynamic range and a negative number and obtaining a spectrum chartappropriately reflecting an intensity of light generated from fineparticles is provided.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a block diagram illustrating a functional configuration of aspectrum analysis apparatus A in accordance with the present technology.

FIG. 2 is a schematic diagram illustrating a configuration of ameasurement unit 10 of the spectrum analysis apparatus A.

FIG. 3 is a graph as a substitute for a drawing illustrating an analysisfunction.

FIG. 4 is a graph as a substitute for a drawing illustrating a spectrumchart in which the horizontal axis represents a PMT channel number andthe vertical axis represents an output value of an analysis function.

FIG. 5 is a graph as a substitute for a drawing illustrating a spectrumchart obtained by measuring a mixed sample in Example 1.

FIG. 6 is a graph as a substitute for a drawing illustrating a spectrumchart reflecting frequency information of beads in the spectrum chartillustrated in FIG. 5 according to a color tone.

FIG. 7 is a graph as a substitute for a drawing illustrating a spectrumchart obtained by measuring a mixed sample and blank beads in Example 1and a spectrum chart of a mixed sample after correction of a backgroundvalue.

FIG. 8 is a graph as a substitute for a drawing illustrating a resultobtained by determining a detection wavelength band of each PMT of a PMTarray in Example 2.

FIG. 9 is a graph as a substitute for a drawing illustrating a resultobtained by calculating the relative sensitivity of each PMT of a PMTarray in Example 2.

FIG. 10 is a graph as a substitute for a drawing illustrating a spectrumchart of fluorescent beads obtained by measurement using a fluorescencespectrophotometer in Example 2.

FIG. 11 is a graph as a substitute for a drawing illustrating a spectrumchart of fluorescent beads, fluorescent particle kit (FPK) 505, obtainedby measurement using a flow cytometer in Example 2 in which a graph (A)illustrates a chart before a correction process, a graph (B) illustratesa chart by a first correction intensity value, and a graph (C)illustrates a chart by a second correction intensity value.

FIG. 12 is a graph as a substitute for a drawing illustrating a spectrumchart of fluorescent beads, FPK 505, obtained by measurement using aflow cytometer in Example 2 in which a graph (A) illustrates a chartbefore a correction process, a graph (B) illustrates a chart by a firstcorrection intensity value, and a graph (C) illustrates a chart by asecond correction intensity value.

FIG. 13 is a graph as a substitute for a drawing illustrating a spectrumchart of fluorescent beads, FPK 528, obtained by measurement using aflow cytometer in Example 2 in which a graph (A) illustrates a chartbefore a correction process, a graph (B) illustrates a chart by a firstcorrection intensity value, and a graph (C) illustrates a chart by asecond correction intensity value.

FIG. 14 is a graph as a substitute for a drawing illustrating a spectrumchart of fluorescent beads, FPK 549, obtained by measurement using aflow cytometer in Example 2 in which a graph (A) illustrates a chartbefore a correction process, a graph (B) illustrates a chart by a firstcorrection intensity value, and a graph (C) illustrates a chart by asecond correction intensity value.

FIG. 15 is a graph as a substitute for a drawing illustrating a spectrumchart of fluorescent beads, FPK 667, obtained by measurement using aflow cytometer in Example 2 in which a graph (A) illustrates a chartbefore a correction process, a graph (B) illustrates a chart by a firstcorrection intensity value, and a graph (C) illustrates a chart by asecond correction intensity value.

FIG. 16 is a graph as a substitute for a drawing illustrating a chart inwhich correction is performed according to a detection wavelength bandwidth of a light-receiving element and the horizontal axis represents adetection wavelength in a spectrum chart obtained by measuring a mixedsample in Example 1.

DESCRIPTION OF EMBODIMENTS

Hereinafter, preferred embodiments of the present technology will bedescribed with reference to the appended drawings. The embodimentsdescribed hereinafter are representative embodiments of the presenttechnology. Thereby, the scope of the present technology is not narrowlyinterpreted. Description will be given in the following order.

1. Configuration of Spectrum Analysis Apparatus

2. Generation of Analysis Data

(1) Analysis Function

(2) Background Correction

(3) Correction according to Detection Wavelength Band Width and RelativeSensitivity of Light-Receiving Element

<Calculation of First Correction Intensity Value>

<Calculation of Second Correction Intensity Value>

3. Data Display

4. Program for Spectrum Analysis and Spectrum Chart Display

1. Configuration of Spectrum Analysis Apparatus

FIG. 1 is a block diagram illustrating a functional configuration of aspectrum analysis apparatus A in accordance with the present technology.In addition, FIG. 2 is a schematic diagram illustrating a configurationof a measurement unit 10 of the spectrum analysis apparatus A.Hereinafter, an example in which the spectrum analysis apparatus A isconfigured as a spectral flow cytometer will be described.

The spectrum analysis apparatus A includes a measurement unit 10 thatdetects fluorescence emitted from fine particles by radiating laserlight to the fine particles, converts an intensity of the detectedfluorescence into an electrical signal, and outputs the electricalsignal as measurement data, a central processing unit (CPU) 20, a memory30, and a hard disk (storage unit) 40. In the spectrum analysisapparatus A, the CPU 20, the memory 30, and the hard disk (storage unit)40 constitute a processing unit. In addition, the spectrum analysisapparatus A includes a mouse 51, a keyboard 52, and a display unit 60configured to include a display 61 and a printer 62 as user interfaces.

The measurement unit 10 can have the same configuration as a fineparticle measurement apparatus of the related art. Specifically, themeasurement unit 10 includes an irradiation system thatcondenses/radiates laser light from a light source 101 to fine particlesP and a detection system including a spectral element 102 thatspectrally separates fluorescence emitted from the fine particles P anda light-receiving element array 103 that detects the spectrallyseparated light. In the spectrum analysis apparatus A, the fineparticles P are arranged in one row inside a flow path formed within aflow cell or on a microchip and flow through the flow path.

The irradiation system includes a condensing lens forcondensing/radiating laser light to the fine particles P, a dichroicmirror, a band pass filter, and the like (not illustrated) in additionto the light source 101. The light source 101 may be a combination oftwo or more light sources that emit light having different wavelengths.In this case, positions in which two or more pieces of laser light areirradiated to the fine particles P may be the same or different. Inaddition, the detection system may include a condensing lens or the like(not illustrated) for condensing fluorescence generated from the fineparticles P and performing light guiding to the spectral element 102.Here, an example of a configuration using a PMT array in which PMTs of32 channels having different detection wavelength bands are arranged inone dimension is illustrated as the light-receiving element array 103.An array of a plurality of independent detection channels havingdifferent detection wavelength bands such as photodiodes or 2Dlight-receiving elements such as CCDs, or CMOSs can also be used in thelight-receiving element array 103.

In the spectrum analysis apparatus A, the measurement unit 10 may bealso configured to detect light generated from the fine particles Paccording to the radiation of laser light, for example, forwardscattered light, lateral scattered light, and scattered light ofRayleigh scattering, Mie scattering, or the like, in addition to thefluorescence.

2. Generation of Analysis Data (1) Analysis Function

The CPU 20 and the memory 30 operate in cooperation with an operatingsystem (OS) 42 and a program 41 for a spectrum analysis and a spectrumchart display stored in the hard disk 40, and generates analysis datausing an analysis function from measurement data including an intensityvalue of fluorescence output from the measurement unit 10. The analysisdata is generated using the analysis function in which a linear functionand a logarithmic function are included as function elements and theintensity value is set as a variable from the measurement data.

Here, an n-th intensity value obtained by a PMT of channel k among PMTsof channels 1 to 32 is defined as I[k,n]. In addition, the analysisfunction is defined as F(x) (where x is a variable). In this case, anoutput value of the analysis data is obtained by F(I[k,n]).

In FIG. 3, the analysis function F(x) is illustrated. In the analysisfunction F(x), the linear function is set as a main function element fordata in which the intensity value I is small, and the logarithmicfunction is set as the main function element for data in which theintensity value I is large. In other words, in the analysis functionF(x), an element of the linear function is more strongly applied to thedata in which the intensity value I is small, and an element of thelogarithmic function is more strongly applied to the data in which theintensity value I is large.

It is possible to apply a well-known function of the related art such asa function based on a biexponential technique or a Logicle function tothe analysis function F(x) (see NPL1). More conveniently, a function inwhich the linear function is set as a function element for a measurementdata region RL in which the intensity value I is less than apredetermined value Ia, and the logarithmic function is set as afunction element for a measurement data region RH in which the intensityvalue I is greater than the predetermined value Ia can be used as theanalysis function F(x). More conveniently, as the analysis functionF(x), the linear function can applied to a measurement data region RL inwhich the intensity value I is less than the predetermined value Ia, andthe logarithmic function can be applied to a measurement data region RHin which the intensity value I is greater than the predetermined valueIa.

In this case, it is only necessary to apply one of the linear functionand the logarithmic function as a function element for the intensityvalue Ia serving as a boundary value. In addition, it is desirable thatthe analysis function F(x) be continuous in a boundary between themeasurement data region RL and the measurement data region RH, andslopes of the analysis function F(x) in the measurement data region RLand the measurement data region RH be consistent in the boundary valueIa.

According to conversion of measurement data by the analysis functionF(x), a wide dynamic range utilizing characteristics of the logarithmicfunction can be provided and simultaneously analysis data including anegative value can also be obtained according to characteristics of thelinear axis. The boundary value Ia can be arbitrarily set as long as anadvantageous effect in accordance with the present technology isexhibited, and, for example, can be set according to the methoddisclosed in the above-described NPL 1.

(2) Background Correction

The processing unit corrects a background value using analysis dataobtained by measuring fine particles (a control sample) for negativecontrol such as a cell (unlabeled cell), which is not labeled by afluorochrome, when analysis data is generated. The background value iscorrected by subtracting a measurement data value of the control samplefrom measurement data of a measurement sample.

An intensity value of an n-th control sample obtained by a PMT ofchannel k among the PMTs of channels 1 to 32 is defined as I_(o)[k,n],and the intensity value of the measurement sample is defined as I[k,n].In this case, the background value is corrected by subtracting theintensity value I_(o)[k,n] of the control sample from the intensityvalue I[k,n] of the measurement sample, that is, by calculating a valueof (I[k,n]−I_(o)[k,n]).

It is preferable to generate the above-described analysis data using theanalysis function F(x) from the measurement data (I[k,n]−I_(o)[k,n])after the subtraction.

(3) Correction According to Detection Wavelength Band Width and RelativeSensitivity of Light-Receiving Element

In addition, the processing unit configured to include the CPU 20, thememory 30, and the hard disk 40 performs a correction process ofcalculating a first correction intensity value by correcting anintensity value of fluorescence according to detection wavelength widthof each of light-receiving elements (here, PMTs of channels 1 to 32).Further, the processing unit performs a correction process ofcalculating a second correction intensity value by correcting the firstcorrection intensity value using sensitivity data of each PMT.

<Calculation of First Correction Intensity Value>

The calculation of the first correction intensity value is carried outby dividing an intensity value of fluorescence acquired by each PMT by adetection wavelength band width of each PMT.

Specifically, an intensity value of an n-th control sample obtained by aPMT of channel k among the PMTs of channels 1 to 32 is defined asI[k,n], a detection lower-limit wavelength of a PMT of channel k is setas L[k] and a detection lower-limit wavelength is set as H[k]. In thiscase, the first correction intensity value J₁[k, n] is calculated by thefollowing expression. Here, k indicates an integer of 1 to 32.J₁[k,n]=I[k,n]/(H[k]−L[k])

When an optical system of the measurement unit 10 including the spectralelement 102 has non-linearity, wavelength band widths of light detectedby the PMTs of channels 1 to 32 become different between the PMTs (seeFIG. 8 given later). Thus, the intensity value of fluorescence acquiredby each PMT is relatively large in a channel in which a detectionwavelength width is wide and relatively small in a channel in which thedetection wavelength with is narrow, and distortion occurs in a spectralshape.

It is possible to compensate for the distortion of the spectral shapedue to the non-linearity of the above-described optical system in thefirst correction intensity value obtained by dividing the intensityvalue of fluorescence acquired by each PMT by the detection wavelengthband width of each PMT.

The detection wavelength width (H[k]−L[k]) of each PMT is uniquelydetermined by types or layouts of optical elements such as the spectralelement 102, the condensing lens, the dichroic mirror, and the band passfilter constituting the measurement unit 10 (see FIG. 8 given later).Thus, in the step in which an apparatus design including the selectionand layouts of the optical elements has been completed, it is possibleto calculate the first correction intensity value from the intensityvalue of fluorescence acquired by each PMT by acquiring a detectionwavelength width of each PMT.

<Calculation of Second Correction Intensity Value>

The calculation of the second correction intensity value is carried outby dividing the first correction intensity value in each PMT by relativesensitivity of each PMT.

Specifically, the relative sensitivity of a PMT of channel k among thePMTs of channels 1 to 32 is set as S[k]. In this case, the secondcorrection intensity value J₂[k,n] is calculated by the followingexpression.

J ₂ [k,n]=J ₁ [k,n]/S[k]

Here, for the relative sensitivity, an intensity value obtained in eachchannel by radiating light of the same intensity and the same wavelengthto the PMT is indicated by a relative value to an intensity value of achannel in which the highest intensity value has been obtained. Therelative sensitivity can be pre-calculated from sensitivity datarecording an electrical signal amount output from each channel whenlight of the same intensity and the same wavelength has been radiated tothe PMT. Both a sensitivity difference inherent in each PMT and asensitivity difference (gain) set by a user to each PMT are reflected inthe sensitivity data. The gain can be appropriately adjusted by changinga setting value such as an applied voltage.

The sensitivities of the PMTs of channels 1 to 32 are different betweenthe PMTs according to an individual difference of the PMT and a gainsetting difference (see FIG. 9 given later). Thus, the intensity valueof fluorescence acquired by each PMT is relatively large in a channel inwhich the sensitivity is high and relatively small in a channel in whichthe sensitivity is low, and distortion occurs in a spectral shape.

It is possible to compensate for the distortion of the spectral shapedue to a sensitivity difference between the above-describedlight-receiving elements in the second correction intensity valueobtained by dividing the first correction intensity value of each PMT bythe relative sensitivity of each PMT.

It is preferable to generate the above-described analysis data using theanalysis function F(x) from measurement data (J₁[k,n] or J₂[k,n]) aftera correction process for the first correction intensity value or asecond measurement intensity value. The correction process for the firstcorrection intensity value or the second measurement intensity value isnot limited to a method to be performed for measurement data beforeconversion by the analysis function F(x) as described above, and can beperformed for analysis data after the conversion by the analysisfunction F(x).

3. Data Display

The processing unit generates a spectrum chart in which one axisrepresents a value corresponding to a detection wavelength band and theother axis represents an output value of an analysis function, andcauses the display unit 60 to display the spectrum chart. The spectrumchart can have the horizontal axis representing a channel number or adetection wavelength of the PMT as a value corresponding to a detectionwavelength band and the vertical axis representing an output value ofthe analysis function (see (C) of FIGS. 5 and 16 given later).

The spectrum chart in which the horizontal axis represents the channelnumber of the PMT and the vertical axis represents the output value ofthe analysis function is illustrated in FIG. 4. An n-th intensity valueobtained by the PMT of channel k is set as I[k,n], and the output valueis set as F(I[k,n]). The number of fine particles (an event count ordensity) included in an output value range that is greater than or equalto V_(i) and less than V_(i+1) is calculated, and a region correspondingto channel k and the intensity V_(i) to V_(i+1) is colored with a colortone corresponding to a value of the calculation result. It is possibleto create and display the spectrum chart illustrated in the drawing byiterating this procedure for each channel and the output value range.Information (frequency information) regarding the number of fineparticles is obtained by performing a multicolor display of the spectrumchart according to a hue, saturation, and/or brightness reflecting theinformation. The conversion of the frequency information into the hue,saturation, and/or brightness can be performed by a technique well knownin the related art (see the examples).

In the spectrum chart, it is possible to display a wide dynamic rangeutilizing characteristics of a logarithmic function according to theconversion of measurement data by the analysis function F(x), and alsoexpress a negative output value according to characteristics of thelinear axis. In addition, it is possible to solve a problem in that thespectrum is extracted with unreasonably high dispersion in a region inwhich the intensity value is small.

Further, when the above-described background correction for the analysisdata value of the vertical axis has been performed in the spectrumchart, it is possible to display the spectrum even when measurement data(I[k,n]−I_(o)[k,n]) after subtraction becomes a negative value.

It is preferable to correct the analysis data value of the vertical axisaccording to the detection wavelength band width and the relativesensitivity of the above-described light-receiving element. Thereby, itis possible to display a chart in which the distortion of the spectralshape due to the non-linearity of the optical system of the apparatusand the sensitivity difference between the light-receiving elements hasbeen compensated for. When the correction according to the detectionwavelength band width or the like of the light-receiving element isperformed, the horizontal axis of the spectrum chart represents thedetection wavelength of the PMT (see (B)/(C) of FIGS. 11 to 15 givenlater).

In the spectrum chart, it is possible to display the intensity value bya mean value, a standard error, a median value, or a statisticalnumerical value of a quartile point or the like based on the number offine particles (an event count or a density) detected in a predeterminedfluorescence intensity value in a predetermined detection wavelength(see FIG. 12 given later). Further, the spectrum chart can also bedisplayed as a three-dimensional (3D) graph to which a coordinate axisrepresenting an event count has been added. This 3D graph can bedisplayed according to a pseudo 3D display.

4. Program for Spectrum Analysis and Spectrum Chart Display

The spectrum analysis program and the spectrum chart display program inaccordance with the present technology execute the steps of generatinganalysis data and displaying data in the above-described spectrumanalysis apparatus.

The program (see reference numeral 41 in FIG. 1) is stored/retained inthe hard disk 40, and loaded into the memory 30 under control of the CPU20 and the OS 42. The program executes a process of generating analysisdata and displaying data. The program can be recorded on acomputer-readable recording medium. The recording medium is notparticularly limited as long as the recording medium is acomputer-readable recording medium. Specifically, for example, aflexible disk or a disk-shaped recording medium such as a compact discread only memory (CD-ROM) is used. In addition, a tape recording mediumsuch as a magnetic tape may be used.

Additionally, the present technology may also be configured as below.

(1) A spectrum analysis apparatus including:

a processing unit configured to generate analysis data using an analysisfunction in which a linear function and a logarithmic function areincluded as function elements and an intensity value is set as avariable from measurement data including the intensity value of lightacquired by detecting the light from a measurement target object using aplurality of light-receiving elements having different detectionwavelength bands.

(2) The spectrum analysis apparatus according to (1), including:

a display unit configured to display the analysis data in a spectrumchart in which one axis represents a value corresponding to thedetection wavelength band and the other axis represents an output valueof the analysis function.

(3) The spectrum analysis apparatus according to (2), wherein theprocessing unit generates the analysis data by applying, as the analysisfunction, a function in which the linear function is set as a mainfunction element for data in which the intensity value is less than apredetermined value among the measurement data and a function in whichthe logarithmic function is set as the main function element for data inwhich the intensity value is greater than the predetermined value amongthe measurement data.

(4) The spectrum analysis apparatus according to (2) or (3), wherein theprocessing unit generates the analysis data by more strongly applying,as the analysis function, an element of the linear function to data inwhich the intensity value is less than a predetermined value among themeasurement data and an element of the logarithmic function to data inwhich the intensity value is greater than a predetermined value amongthe measurement data.

(5) The spectrum analysis apparatus according to (4), wherein thedisplay unit displays the spectrum chart in which an axis of the outputvalue is set as a logarithmic axis in a region in which the output valueis greater than a predetermined value and set as a linear axis in aregion in which the output value is less than the predetermined value.

(6) The spectrum analysis apparatus according to any one of (2) to (5),

wherein the processing unit generates analysis data corrected using theanalysis function in which the linear function and the logarithmicfunction are included as the function elements and the intensity valueis set as the variable, after subtracting measurement data including anintensity value obtained by detecting light from a control sample usinga plurality of light-receiving elements having different detectionwavelength bands from measurement data including an intensity valueobtained by detecting light from a measurement sample using a pluralityof light-receiving elements having different detection wavelength bands,and wherein the display unit displays the corrected analysis data in thespectrum chart.

(7) The spectrum analysis apparatus according to any one of (1) to (7),wherein the measurement data includes the intensity value of lightdetected and obtained by a light-receiving element array in which aplurality of light-receiving elements having different detectionwavelength bands are arranged to spectrally separate the light from ameasurement target object.

(8) The spectrum analysis apparatus according to any one of (1) to (7),wherein the processing unit corrects the intensity value according to adetection wavelength band width of each light-receiving element andgenerates the analysis data.

(9) The spectrum analysis apparatus according to any one of (1) to (8),

wherein the measurement target object is fine particles, and

wherein optical characteristics of the fine particles are displayed inthe spectrum chart.

(10) The spectrum analysis apparatus according to any one of (1) to (9),wherein the display unit performs a multicolor display of the spectrumchart.

(11) The spectrum analysis apparatus according to (9) or (10), whereinthe display unit performs the multicolor display of the spectrum chartaccording to a hue, saturation, and/or brightness reflecting frequencyinformation of the fine particles.

Example 1 1. Spectrum Chart Generation and Background Correction

A prototype spectral flow cytometer equipped with the measurement unitof the configuration illustrated in FIG. 2 was made. As the lightsource, a laser diode with a wavelength of 488 nm and a laser diode witha wavelength of 638 nm were used. In addition, as the spectral element,a prism array in which a plurality of prisms are combined was used. Asthe light-receiving element array, a PMT array of 32 channels was used.Fluorescence of a wavelength 500 nm to 800 nm was spectrally detected.

Using this apparatus, mixed samples of fluorescent beads, FPK 505, FPK528, and FPK 549, acquired from Sherotech Inc. and unlabeled negativecontrol beads (blank beads) were measured. After an appropriatepopulation was extracted according to gating using statistical softwareR (http://www.R-project.org/) for the acquired data, a spectrum chartwas displayed.

The results are illustrated in FIG. 5. A graph (A) illustrates a chartin which the vertical axis represents the intensity value I as thelogarithmic axis, and a graph (B) illustrates a chart in which thelinear axis represents the intensity value I. A graph (C) illustrates aspectrum chart in which the vertical axis is set as the logarithmic axisin a region in which the intensity value I is greater than 10,000, andset as the linear axis in a region in which the intensity value I isless than 10,000. The horizontal axis represents a channel number of thePMT. Here, bead frequency information is indicated according to thegradation of the spectrum chart.

In the logarithmic-axis chart illustrated in the graph (A), dispersionof the spectrum of (dark) beads in which a fluorescent level is low isdisplayed to be very large. In addition, in the linear-axis chartillustrated in the graph (B), dispersion of the spectrum of (bright)beads in which a fluorescent level is high is displayed to be very largeand it is difficult to discriminate a spectral shape of dark beads. Onthe other hand, in the hybrid-axis chart illustrated in the graph (C),the spectrum of dark beads is not extremely wide, a sharp shape in whichdispersion is low can be displayed, and spectral shapes of three typesof beads for a wide dynamic range can be displayed in a state in whichthey are capable of being clearly discriminated.

In addition, FIG. 6 illustrates a spectrum chart reflecting beadfrequency information according to a color tone in the chart illustratedin the graph (C) of FIG. 5. A graph (A) is a chart obtained byperforming conversion into a hue, saturation, and/or brightness of thefrequency information using a “rainbow” function of the statisticalsoftware R. A graph (B) is a chart obtained by performing conversioninto a hue, saturation, and/or brightness of the frequency informationusing a “topo. colors” function of the statistical software R. A graph(C) is a chart obtained by performing conversion into a hue, saturation,and/or brightness of the frequency information using a “cm. colors”function of the statistical software R. A graph (D) is a chart obtainedby performing conversion into a hue, saturation, and/or brightness ofthe frequency information using a “terrain. colors” function of thestatistical software R. A graph (E) is a chart obtained by performingconversion into a hue, saturation, and/or brightness of the frequencyinformation using a “heat. colors” function of the statistical softwareR. A graph (F) is a chart obtained by performing conversion into a hue,saturation, and/or brightness of the frequency information using a“grey” function of the statistical software R. In these spectrum charts,drawings of the measurement results of the PMTs of channels 20 to 32 areomitted.

FIG. 7 illustrates results obtained by performing background correctionin which measurement data of blank beads is subtracted from themeasurement data of a mixed sample. Graphs (A) to (C) illustrate chartsin which the vertical axis represents the intensity values I and I_(o)as the logarithmic axis, and graphs (D) to (F) illustrate spectrumcharts in which the vertical axis is set as the logarithmic axis in aregion in which the intensity value I or the intensity value (I−I_(o))after subtraction is greater than 10,000, and set as the linear axis ina region in which the intensity value I or the intensity value (I−I_(o))is less than 10,000. In addition, the graphs (A) and (D) illustratespectrum charts of mixed samples, the graphs (B) and (D) illustratespectrum charts of blank beads, and the graphs (C) and (F) illustratespectrum charts of mixed samples after background correction.

In the chart illustrated in the graph (F), it is possible to clearlyrecognize a spectral shape of dark beads as compared to the chartillustrated in the graph (C).

As seen from the above-described results, according to the spectrumanalysis apparatus in accordance with the present technology, a widedynamic range including a negative number can be provided and a spectrumchart appropriately reflecting an intensity of light generated from fineparticles can be displayed.

Example 2 2. Correction According to Detection Wavelength Band Width andRelative Sensitivity of Light-Receiving Element

A graph determining a detection wavelength band in the prototypeapparatus is illustrated in FIG. 8. In the graph, “X” denotes adetection lower-limit wavelength L[k] of the PMT of each channel, and“O” denotes a detection upper-limit wavelength H[k]. Here, k denotes aninteger of 1 to 32. The detection wavelength band width (H[k]−L[k]) ofeach PMT is recognized to be as wide as in the PMT of a long-wavelengthside. In the PMTs after and before channel 21 that detects fluorescencearound a wavelength of 638 nm, the detected fluorescence is also limitedby an optical filter that prevents the leakage of laser light from alight source with the wavelength of 638 nm.

In addition, a graph obtained by calculating the relative sensitivity ofeach PMT is illustrated in FIG. 9. The relative sensitivity is indicatedby a relative value when the intensity value of channel 32 in which thehighest intensity value has been obtained among the intensity valuesobtained by the channels by radiating light of the same intensity andthe same wavelength to the PMTs is set to 1.

Initially, a fluorescence spectrum of commercially available fluorescentbeads was measured using an F-4500 fluorescence spectrophotometer(Hitachi High-Technologies Corporation). As the fluorescent beads, fourtypes of FPK 505, FPK 528, FPK 549, and FPK 667 acquired from SherotechInc. were used. The obtained spectrum chart (standard spectrum chart) isillustrated in FIG. 10. A graph (A) illustrates the fluorescencespectrum of FPK 505, a graph (B) illustrates the fluorescence spectrumof FPK 528, a graph (C) illustrates the fluorescence spectrum of FPK549, and a graph (D) illustrates the fluorescence spectrum of FPK 667.The horizontal axis represents a fluorescent wavelength (500 to 800 nm),and the vertical axis represents a fluorescent intensity value(logarithmic indication). An excitation wavelength of the laser light isa wavelength of 488 nm in the graphs (A) to (C) and a wavelength of 638nm in the graph (D).

Next, the fluorescence spectrum of fluorescent beads was measured usingthe prototype apparatus. The obtained spectrum chart is illustrated inFIGS. 11 to 15. FIGS. 11 and 12 illustrate charts of FPK 505, FIG. 13illustrates a chart of FPK 528, FIG. 14 illustrates a chart of FPK 549,and FIG. 15 illustrates a chart of FPK 667. In FIG. 11, the event countin each channel is displayed in the color of a spectrum. In addition, inFIG. 12, the intensity value is displayed by a mean value (indicated bythe solid line) and Mean Value±Standard Deviation (indicated by thedotted line) based on the event count.

In graphs (A) of FIGS. 11 to 15, spectrum charts in which the horizontalaxis represents a channel number and the vertical axis represents alogarithm of an intensity value I[k] (where k denotes an integer of 1 to32) of fluorescence acquired in each channel are illustrated.

The spectral shapes illustrated in the spectrum charts of the graphs (A)of FIGS. 11 to 15 are clearly different from the spectral shape of thestandard spectrum chart illustrated in FIG. 10. This indicates thatdistortion occurs in the spectral shape due to a measurement errorcaused by the optical system of the apparatus and a sensitivitydifference between the light-receiving elements in the fluorescencespectrum directly using the intensity value I[k] of fluorescenceacquired by the PMT.

In graphs (B) of FIGS. 11 to 15, spectrum charts in which the horizontalaxis represents a detection wavelength and the vertical axis representsa logarithm of the first correction intensity value J₁[k] (where kdenotes an integer of 1 to 32) of a fluorescence intensity valueacquired in each channel are illustrated. The first correction intensityvalue J₁[k] was obtained by dividing the intensity value I[k] offluorescence acquired by each PMT by the detection wavelength band width(H[k]−L[k]) of each PMT illustrated in FIG. 8. More specifically, thespectrum chart was created by dividing an n-th intensity value I[k,n]acquired by the PMT of channel k by the detection wavelength band width(H[k]−L[k]) of the PMT to obtain the first correction value J₁[k] anddrawing a distribution of J₁[k,n] in a range of L[k] to H[k] of thehorizontal axis.

Spectral shapes illustrated in the spectrum charts of the graphs (B) ofFIGS. 11 to 15 are substantially consistent with the spectral shape ofthe standard spectrum chart illustrated in FIG. 10. This indicates thatthe distortion of the spectral shape can be corrected by compensatingfor the measurement error due to the non-linearity of the optical systemof the apparatus according to a correction process of dividing theintensity value I[k] of fluorescence acquired by each PMT by thedetection wavelength band width (H[k]−L[k]) of each PMT.

In graphs (C) of FIGS. 11 to 15, spectrum charts in which the horizontalaxis represents a detection wavelength and the vertical axis representsa logarithm of the second correction intensity value J₂[k] (where kdenotes an integer of 1 to 32) of a fluorescence intensity valueacquired in each channel are illustrated. The second correctionintensity value J₂[k] was obtained by dividing the first correctionintensity value J₁[k] by the relative sensitivity S[k] of each PMTillustrated in FIG. 9.

Spectral shapes illustrated in the spectrum charts of the graphs (C) ofFIGS. 11 to 15 are consistent with the spectral shape of the standardspectrum chart illustrated in FIG. 10. In particular, although thedistortion of the spectral shape assumed to be caused by a sensitivitydifference of the PMT in a region of around a wavelength of 500 nm isviewed in the spectrum chart based on the first correction intensityvalue J₁[k] of (B) of FIGS. 11 to 15, the distortion is corrected in thespectrum charts based on the second correction intensity value J₂[k] ofthe graphs (C) of FIGS. 11 to 15. This indicates that the distortion ofthe spectral shape can be corrected by compensating for the measurementerror due to the sensitivity difference between the light-receivingelements according to a correction process of dividing the firstcorrection intensity value J₁[k] by the relative sensitivity S[k] ofeach PMT.

FIG. 16 illustrates the spectrum chart in which the data obtained inExample 1 is corrected according to a detection wavelength band widthand the horizontal axis represents a detection wavelength. In thehybrid-axis chart illustrated in a graph (C), the spectrum of dark beadsis not extremely wide, a sharp shape in which dispersion is low can bedisplayed, and spectral shapes of three types of beads for a widedynamic range can be displayed in a state in which they are capable ofbeing clearly discriminated.

It should be understood by those skilled in the art that variousmodifications, combinations, sub-combinations and alterations may occurdepending on design requirements and other factors insofar as they arewithin the scope of the appended claims or the equivalents thereof

INDUSTRIAL APPLICABILITY

In the spectrum analysis apparatus in accordance with the presenttechnology, it is possible to display a wide dynamic range and anegative number and obtain a spectrum chart appropriately reflectingoptical characteristics of a measurement target object. The spectrumanalysis apparatus in accordance with the present technology can beappropriately applied to a fine particle measurement apparatus foranalyzing optical characteristics of fine particles of a cell and thelike in further detail, particularly, a spectral flow cytometer.

REFERENCE SIGNS LIST

-   A Spectrum analysis apparatus-   P Fine particles-   10 Measurement unit-   101 Light source-   102 Spectral element-   103 Light-receiving element array-   20 CPU-   30 Memory-   40 Hard disk (storage unit)-   41 Fluorescence intensity correction program-   420S-   51 Mouse-   52 Keyboard-   60 Display unit-   61 Display-   62 Printer

1. A spectrum analysis apparatus comprising: a processing unitconfigured to generate analysis data using an analysis function in whicha linear function and a logarithmic function are included as functionelements and an intensity value is set as a variable from measurementdata including the intensity value of light acquired by detecting thelight from a measurement target object using a plurality oflight-receiving elements having different detection wavelength bands. 2.The spectrum analysis apparatus according to claim 1, comprising: adisplay unit configured to display the analysis data in a spectrum chartin which one axis represents a value corresponding to the detectionwavelength band and the other axis represents an output value of theanalysis function.
 3. The spectrum analysis apparatus according to claim2, wherein the processing unit generates the analysis data by applying,as the analysis function, a function in which the linear function is setas a main function element for data in which the intensity value is lessthan a predetermined value among the measurement data and a function inwhich the logarithmic function is set as the main function element fordata in which the intensity value is greater than the predeterminedvalue among the measurement data.
 4. The spectrum analysis apparatusaccording to claim 2, wherein the processing unit generates the analysisdata by more strongly applying, as the analysis function, an element ofthe linear function to data in which the intensity value is less than apredetermined value among the measurement data and an element of thelogarithmic function to data in which the intensity value is greaterthan a predetermined value among the measurement data.
 5. The spectrumanalysis apparatus according to claim 4, wherein the display unitdisplays the spectrum chart in which an axis of the output value is setas a logarithmic axis in a region in which the output value is greaterthan a predetermined value and set as a linear axis in a region in whichthe output value is less than the predetermined value.
 6. The spectrumanalysis apparatus according to claim 5, wherein the processing unitgenerates analysis data corrected using the analysis function in whichthe linear function and the logarithmic function are included as thefunction elements and the intensity value is set as the variable, aftersubtracting measurement data including an intensity value obtained bydetecting light from a control sample using a plurality oflight-receiving elements having different detection wavelength bandsfrom measurement data including an intensity value obtained by detectinglight from a measurement sample using a plurality of light-receivingelements having different detection wavelength bands, and wherein thedisplay unit displays the corrected analysis data in the spectrum chart.7. The spectrum analysis apparatus according to claim 6, wherein themeasurement data includes the intensity value of light detected andobtained by a light-receiving element array in which a plurality oflight-receiving elements having different detection wavelength bands arearranged to spectrally separate the light from a measurement targetobject.
 8. The spectrum analysis apparatus according to claim 7, whereinthe processing unit corrects the intensity value according to adetection wavelength band width of each light-receiving element andgenerates the analysis data.
 9. The spectrum analysis apparatusaccording to claim 8, wherein the measurement target object is fineparticles, and wherein optical characteristics of the fine particles aredisplayed in the spectrum chart.
 10. The spectrum analysis apparatusaccording to claim 9, wherein the display unit performs a multicolordisplay of the spectrum chart.
 11. The spectrum analysis apparatusaccording to claim 10, wherein the display unit performs the multicolordisplay of the spectrum chart according to a hue, saturation, and/orbrightness reflecting frequency information of the fine particles.
 12. Afine particle measurement apparatus comprising: the spectrum analysisapparatus according to claim
 11. 13. The fine particle measurementapparatus according to claim 12, wherein the fine particle measurementapparatus is a spectral flow cytometer.
 14. A spectrum analysis methodcomprising: generating analysis data using an analysis function in whicha linear function and a logarithmic function are included as functionelements and an intensity value is set as a variable from measurementdata including the intensity value of light acquired by detecting thelight from a measurement target object using a plurality oflight-receiving elements having different detection wavelength bands.15. A spectrum-chart displaying method, comprising: displaying analysisdata generated using an analysis function in which a linear function anda logarithmic function are included as function elements and anintensity value is set as a variable from measurement data including theintensity value of light acquired by detecting the light from ameasurement target object using a plurality of light-receiving elementshaving different detection wavelength bands in a spectrum chart in whichone axis represents a value corresponding to the detection wavelengthband and the other axis represents an output value of the analysisfunction.
 16. A spectrum analysis program for executing: generatinganalysis data using an analysis function in which a linear function anda logarithmic function are included as function elements and anintensity value is set as a variable from measurement data including theintensity value of light acquired by detecting the light from ameasurement target object using a plurality of light-receiving elementshaving different detection wavelength bands.
 17. A spectrum-chartdisplaying program for executing: displaying analysis data generatedusing an analysis function in which a linear function and a logarithmicfunction are included as function elements and an intensity value is setas a variable from measurement data including the intensity value oflight acquired by detecting the light from a measurement target objectusing a plurality of light-receiving elements having different detectionwavelength bands in a spectrum chart in which one axis represents avalue corresponding to the detection wavelength band and the other axisrepresents an output value of the analysis function.